The notch and TGF-β signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma

Background

Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown.

Methodology/Principal Findings

We show that inhibition of endogenous Notch signaling modulates TGF-β dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-β pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-β signaling activity (two-sided Student''s t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-β1 induced TGF-β signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells.

Conclusion

An extensive cross-talk between the Notch and TGF-β signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.     

Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume,morphology, rheology and hemolysis

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis.We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry.Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3–36-fold) and hemolysis (1–44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43–67%).The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis.For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure.HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60–80%. HlyA-RBCs showed an acute 1.3–2.2-fold increase of Ca²⁺i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media.Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.     

Regulation of gene expression,growth,and cell survival by IL—4：Contribution of multiple signaling pathways

Interleukin-4 is a cytokine produced by activated T cells,mast cells,and basophils that elicits many important biological responses[1](see Tab 1).These responses range from the regulation of helper T cell differentiation[2] and the production of IgE[3] to the regulation of the adhesive properties of endothelial cells via VCAM-1[4],In keeping with these diverse biological effects,high-affinity binding sites for IL-4(Kd 20 to 300pM) have been detected on many hematopoietic and non-hematopoietic cell types at levels ranging from 50 to 5000 sites per cell[5].This review will focus on the discrete signal transduction pathways activated by the IL-4 recxeptor and the coordination of these individual pathways in the regulation of a final biological outcome.     

A novel approach to evaluate ELISA antibody coverage of host cell proteins—combining ELISA-based immunocapture and mass spectrometry

Monitoring host cell proteins (HCPs) is one of the most important analytical requirements in production of recombinant biopharmaceuticals to ensure product purity and patient safety. Enzyme-linked immunosorbent assay (ELISA) is the standard method for monitoring HCP clearance. It is important to validate that the critical reagent of an ELISA, the HCP antibody, covers a broad spectrum of the HCPs potentially present in the purified drug substance. Current coverage methods for assessing HCP antibody coverage are based on 2D-Western blot or immunoaffinity-purification combined with 2D gel electrophoresis and have several limitations. In the present study, we present a novel coverage method combining ELISA-based immunocapture with protein identification by liquid chromatography–tandem mass spectrometry (LC–MS/MS): ELISA-MS. ELISA-MS is used to accurately determine HCP coverage of an early process sample by three commercially available anti-Escherichia coli HCP antibodies, evading the limitations of current methods for coverage analysis, and taking advantage of the benefits of MS analysis. The results obtained comprise a list of individual HCPs covered by each HCP antibody. The novel method shows high sensitivity, high reproducibility, and enables tight control of nonspecific binding through inclusion of a species-specific isotype control antibody. We propose that ELISA-MS will be a valuable supplement to existing coverage methods or even a replacement. ELISA-MS will increase the possibility of selecting the best HCP ELISA, thus improving HCP surveillance and resulting in a final HCP profile with the lowest achievable risk. Overall, this will be beneficial to both the pharmaceutical industry and patient safety.     

Theoretical studies on the interaction of proteins and nucleic acid: II. The binding of α-helix to B-DNA

Interactions between B-DNA and homopolymeric α-helices of glycine, alanine, serine, asparagine and aspartic acid have been studied theoretically. The complexation energy has been minimised taking into account the interactions between DNA and the polypeptides as well as the internal energy of the α-helix and the interaction energy of counterions with the complex. The results obtained indicate the important role of strong hydrogen bonds between the peptide side chains and nucleic acid phosphate groups, these bonds being much stronger than specific interactions with the base-pairs. The formation of these structural bonds depends on the size of the α-helix, which in turn determines whether bridging across the major groove is possible. The steric role of the methyl group of thymine in orienting the peptide helix and the role of DNA screening cations in complex stabilization are also significant.     

A variant of Saccharomyces cerevisiae pep4 strain with improved oligotrophic proliferation, cell survival and heterologous secretion of α-amylase

A variant of Saccharomyces cerevisiae pep4 strain 20B12, with improved oligotrophic proliferation, cell survival and secretion of heterologous mouse α-amylase, is described. Previously we reported a procedure to enrich NI transformants that are not inhibited by cytotoxic expression of hepatitis B virus surface antigen in the secretion pathway of the protease-A-deficient (pep4) strain. To use the NI cells as a host for heterologous expression, we tried to amend the introduced pYAS/12S vector and obtain a host strain, NI-C, with stable NI phenotype and trp1 marker restored. Southern analysis of genomic DNA of NI-C suggested that the original pYAS/12S was abnormally rearranged and not completely corrected. Further assay showed that the viability and mitotic ability of the NI-C strain were increased. While using the NI-C strain as host for plasmid transformation and heterologous expression of mouse α-amylase, we observed that transformed colonies grew more quickly and secreted more α-amylase than general yeast strains. A further test showed that the NI-C strain was able to use mouse α-amylase as a positive selection marker to form transformed colonies on nitrogen-starved plates that contain starch as the sole carbon source. The results imply that the NI-C variant is an improved pep4 strain that can be used for heterologous expression and for the development of new selective markers in the yeast transformation system. Received: 7 January 1998 / Received last revision: 7 September 1998 / Accepted: 11 October 1998     

The association between RDW and survival of patients with squamous cell carcinoma of the tongue. Simple,cheap and convenient?

AimThe purpose of this study was to evaluate the prognostic impact of red-cell distribution width (RDW) on the overall survival (OS) of patients with squamous cell carcinoma (SCC) of the tongue.BackgroundDevelopment of cancer is connected with an ongoing inflammatory process which is reflected by laboratory indices, such as RDW that can be used as prognostic tools.Material and methodsThe study group consists of 74 consecutive patients treated with radical radiotherapy or chemo-radiotherapy for SSC of the tongue at one institution between 2005−2014. RDW was assessed based on routine blood tests done before the start of the treatment. ROC curve was applied to assess value of RDW in prediction of OS, and a cut-off value for further tests was obtained using the Younden index. The survival analysis was performed using the Kaplan-Meier method, log-rank testing and Cox regression model.ResultsThe AUC for RDW in ROC analysis was 0.703, and the optimal cut-off value was 13.5%. 5-year OS was significantly lower in patients with RDW ≥ 13.5% compared with patients with RDW < 13.5% (67% vs. 26%, p-value = 0.0005). Additionally, high RDW was associated with a greater odds ratio for 5-year OS in a multivariate Cox proportional hazards regression analysis (3.43, 1.62–7.25; p = 0.001).ConclusionOur study demonstrated that pre-treatment RDW ≥ 13,5% is an indicator of poor overall survival in patients with SCC of the tongue. Since RDW is a cheap and convenient marker, usually routinely assessed during complete blood count tests, it could be further used as an additional prognostic tool in patients with tongue cancers.     

The phospholipid code: a key component of dying cell recognition,tumor progression and host–microbe interactions

A significant effort is made by the cell to maintain certain phospholipids at specific sites. It is well described that proteins involved in intracellular signaling can be targeted to the plasma membrane and organelles through phospholipid-binding domains. Thus, the accumulation of a specific combination of phospholipids, denoted here as the ‘phospholipid code'', is key in initiating cellular processes. Interestingly, a variety of extracellular proteins and pathogen-derived proteins can also recognize or modify phospholipids to facilitate the recognition of dying cells, tumorigenesis and host–microbe interactions. In this article, we discuss the importance of the phospholipid code in a range of physiological and pathological processes.     

Mapping through somatic cell hybrids and cDNA probes of protein C to chromosome 2, factor X to chromosome 13, and α1-acid glycoprotein to chromosome 9

Summary The previously unassigned gene coding for the anticoagulatory protein C has been mapped on chromosome 2 using a cDNA probe and genomic blots from a human-hamster somatic cell hybrid panel. The assignments of the genes coding for the coagulation factor X to chromosome 13, and for 1-acid glycoprotein to chromosome 9 have been confirmed using a similar direct approach.     

Sensitive and resistant of the homologous disulfide-bridged proteins α-lactalbumin and lysozyme to attack of hydrogen-atoms,dithiothreitol and trifluoroacetic acid,examined by matrix-assisted laser desorption/ionization mass spectrometry

BackgroundEvolutionarily homologous proteins bovine α-lactoalbumin (αLA) and hen egg-white lysozyme (HEL) are very similar in primary, secondary and tertiary structures involving the location of disulfide-bridges (S–S), and are resistant to the action of hydrolytic enzymes and reagents. It is of interest to examine and compare the difference in backbone cleavage characteristics, by using reductive and hydrolytic reagents.MethodsIn-source decay (ISD) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), reductive treatment of αLA and HEL with dithiothreitol (DTT) and acid hydrolysis with trifluoroacetic acid (TFA) were employed to examine the difference in the backbone cleavage characteristics of αLA and HEL.ResultsThe treatment of αLA and HEL with DTT/AcOHNH₃ resulted in similar cleavage behaviors of the backbone N-Cα and S–S bonds, i.e., the enhancements of the intensity and m/z range of sequence-reflected fragment ions were very similar. However, the treatment of αLA with DTT/TFA resulted in unexpected residue-specific degradation at the peptide bond of the Asp-Xxx, Xxx-Ser/Thr, Gln-Xxx, Xxx-Gly and Gly-Xxx residues, while HEL did not occur such degradation.ConclusionsThe results obtained above indicate that acidic αLA is very sensitive to acidic additive such as TFA, while basic HEL is resistance to acidic additives.General significanceThe study demonstrates the sensitive and resistant of evolutionary homologous proteins αLA and HEL to the acid hydrolysis and these characters come from acidic and basic nature of the proteins.     

Establishment and characterization of HEPFT, a cell line derived from hepatoid carcinoma of the fallopian tube, with special reference to α-fetoprotein, lectin affinity and histogenesis

A cell line designated "HEPFT" was established from a human fallopian tubal hepatoid carcinoma. This line grew well without interruption for 13 months and was subcultivated over 35 times. The cells were spherical and polygonal in shape and showed neoplastic and pleomorphic features such as a bizarre aggregation of chromatin granules, an irregular thickening membrane and multiple large nucleoli. The cells formed epithelial colonies with a jigsaw puzzle-like arrangement and multilayering without contact inhibition. The cells contained moderate to abundant amounts of eosinophilic cytoplasm and were immunohistochemically positive for alpha-fetoprotein. The cells proliferated rapidly, and the population doubling time was about 45 h. The chromosome number showed a wide distribution of aneuploidy. The modal chromosome number was stable in the hyper triploid range and many marker chromosomes were observed. The culture cells produced bile and a large amount of lentil lectin-reactive alpha-fetoprotein. The recently developed bacterial artificial chromosome array comparative genomic hybridization facilitated detailed analysis with high resolution and sensitivity. Different profiles of genomic copy-number abnormalities were demonstrated in various chromosomal regions in HEPFT cells.     

Heterogeneity of mouse primordial germ cells reflecting the distinct status of their differentiation, proliferation and apoptosis can be classified by the expression of cell surface proteins integrin α6 and c-Kit

Primordial germ cells (PGCs) in mouse embryos likely include heterogeneous cells having distinct cellular properties. In the present study, we found that heterogeneity of PGCs can be defined by the expression of integrin α6 and c-Kit. The changes in integrin α6 and c-Kit expression in PGCs were obvious as embryonic development progressed, and the PGCs became a mixture of populations consisting of cells with distinct levels of cell surface protein expression. The changes and heterogeneity of cell surface protein expression mainly reflected asynchronous differentiation of PGCs. Apoptosis of PGCs was biased in populations of c-Kit or integrin α6 negative PGCs at particular developmental stages, suggesting possible linkage between PGC apoptosis and the levels of expression of these cell surface proteins. Histochemical analysis confirmed the heterogeneous expression of c-Kit and integrin α6 in PGCs in embryonic gonads, and revealed that PGCs showing different levels of integrin α6 or c-Kit expression and the apoptotic PGCs were scattered and did not show specific localization within gonads. The present study enables us to analyze and isolate populations of living PGCs showing a distinct status of differentiation, or different properties of proliferation or of cell death in individual embryos, and provides a new strategy to examine the mechanisms of PGC development.     

How heritable is individual susceptibility to death? The results of an analysis of survival data on Danish, Swedish and Finnish twins.

Molecular epidemiological studies confirm a substantial contribution of individual genes to variability in susceptibility to disease and death for humans. To evaluate the contribution of all genes to susceptibility and to estimate individual survival characteristics, survival data on related individuals (eg twins or other relatives) are needed. Correlated gamma-frailty models of bivariate survival are used in a joint analysis of survival data on more than 31,000 pairs of Danish, Swedish and Finnish male and female twins using the maximum likelihood method. Additive decomposition of frailty into genetic and environmental components is used to estimate heritability in frailty. The estimate of the standard deviation of frailty from the pooled data is about 1.5. The hypothesis that variance in frailty and correlations of frailty for twins are similar in the data from all three countries is accepted. The estimate of narrow-sense heritability in frailty is about 0.5. The age trajectories of individual hazards are evaluated for all three populations of twins and both sexes. The results of our analysis confirm the presence of genetic influences on individual frailty and longevity. They also suggest that the mechanism of these genetic influences may be similar for the three Scandinavian countries. Furthermore, results indicate that the increase in individual hazard with age is more rapid than predicted by traditional demographic life tables.     

Fusion of Epstein-Barr virus with epithelial cells can be triggered by αvβ5 in addition to αvβ6 and αvβ8, and integrin binding triggers a conformational change in glycoproteins gHgL

Fusion of herpesviruses with their target cells requires a minimum of three glycoproteins, namely, gB and a complex of gH and gL. Epstein-Barr virus (EBV) fusion with an epithelial cell requires no additional virus glycoproteins, and we have shown previously that it can be initiated by an interaction between integrin αvβ6 or αvβ8 and gHgL. We now report that integrin αvβ5 can also bind to gHgL and trigger fusion. Binding of gHgL to integrins is a two-step reaction. The first step, analyzed by surface plasmon resonance, was fast, with high association and low dissociation rate constants. The second step, detected by fluorescence spectroscopy of gHgL labeled at cysteine 153 at the domain I-domain II interface with the environmentally sensitive probes acrylodan and IANBD, involved a slower conformational change. Interaction of gHgL with neutralizing monoclonal antibodies or Fab' fragments was also consistent with a two-step reaction involving fast high-affinity binding and a subsequent slower conformational change. None of the antibodies bound to the same epitope, and none completely inhibited integrin binding. However, binding of each decreased the rate of conformational change induced by integrin binding, suggesting that neutralization might involve a conformational change that precludes fusion. Overall, the data are consistent with the interaction of gHgL with an integrin inducing a functionally important rearrangement at the domain I-domain II interface.     

Interactions between the tobacco budworm,Heliothis virescens,and the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki: Relationship between length of exposure to the toxin and survival

Larvae of the tobacco budworm, Heliothis virescens, which were fed ad libitum for 24, 48, and 72 hr on a diet treated with various levels of the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki and then transferred to an untreated diet, showed an unexpected capacity to recover from the effects of the toxin, although, as the length of exposure increased, the capacity decreased. Observations on larvae held to emergence indicated that recovery from the toxin was complete. X-ray studies using Ba²⁺ incorporated into the diet showed that, although the toxin paralyzed the midgut of the treated animals, many animals recovered after the toxin was removed, with food once again passing through the gut.     

Parasites as causative agents of human affective disorders? The impact of anti-psychotic, mood-stabilizer and anti-parasite medication on Toxoplasma gondii's ability to alter host behaviour

With increasing pressure to understand transmissible agents, renewed recognition of infectious causation of both acute and chronic diseases is occurring. Epidemiological and neuropathological studies indicate that some cases of schizophrenia may be associated with environmental factors, such as exposure to the ubiquitous protozoan Toxoplasma gondii. Reasons for this include T. gondii's ability to establish persistent infection within the central nervous system, its ability to manipulate intermediate host behaviour, the occurrence of neurological and psychiatric symptoms in some infected individuals, and an association between infection with increased incidence of schizophrenia. Moreover, several of the medications used to treat schizophrenia and other psychiatric disease have recently been demonstrated in vitro to possess anti-parasitic, and in particular anti-T. gondii, properties. Our aim here was thus to test the hypothesis that the anti-psychotic and mood stabilizing activity of some medications may be achieved, or at least augmented, through their in vivo inhibition of T. gondii replication and invasion in infected individuals. In particular we predicted, using the epidemiologically and clinically applicable rat-T. gondii model system, and following a previously described and neurologically characterized 'feline attraction' protocol that haloperidol (an anti-psychotic used in the treatment of mental illnesses including schizophrenia) and/or valproic acid (a mood stabilizer used in the treatment of mental illnesses including schizophrenia), would be, at least, as effective in preventing the development of T. gondii-associated behavioural and cognitive alterations as the standard anti-T. gondii chemotherapeutics pyrimethamine with Dapsone. We demonstrate that, while T. gondii appears to alter the rats' perception of predation risk turning their innate aversion into a 'suicidal' feline attraction, anti-psychotic drugs prove as efficient as anti-T. gondii drugs in preventing such behavioural alterations. Our results have important implications regarding the aetiology and treatment of such disorders.     

The toxicity of bovine α-lactalbumin made lethal to tumor cells is highly dependent on oleic acid and induces killing in cancer cell lines and noncancer-derived primary cells

A complex between α-lactalbumin and oleic acid (C18:1, 9 cis) has been reported to be cytotoxic to cancer cells. We have prepared such complexes and tested their activity against both cancer cell lines and noncancer-derived primary cells. Unexpectedly, some primary cell types were more sensitive to treatment than cancer cell lines. We found the complex to be cytotoxic to all of the tested cells, with a 46-fold difference between the most sensitive and the least sensitive cell type. Oleic acid by itself exhibited a remarkably similar activity. The cell-killing mechanisms of the complex and of oleic acid alone were examined by flow cytometry, testing for apoptosis- and necrosis- inducing activity. The T-cell leukemia-derived Jurkat cells primarily underwent cell death resembling apoptosis, whereas the monocytic leukemia-derived THP1 cells adopted a more necrotic-like cell death. Erythrocytes were sensitive to lysis by the complex and oleic acid. We conclude that oleic acid is cytotoxic by itself and that, in contrast to the literature, a complex of α-lactalbumin and oleic acid has cytotoxic activity against primary cells, as well as cancer cells.